PROJECT SUMMARY In the canonical pathway, TGF?s bind to their type I and II receptors TGF?RII and ALK5, respectively, to activate Smads 2 and 3. It has been shown that Smad3 is essential for articular cartilage maintenance, but the function of Smad2 in vivo is unknown. It has been shown that TGF?RII is not required for chondrogenesis during development, but the function of the type I receptor ALK5 is unknown. This is an important gap in knowledge because ALK5 can activate TGF? pathways independently of TGF?RII. Moreover, ALK5 can interact with the type I BMP receptor ALK1, enabling TGF?s to activate BMP pathways. We found that loss of ALK5 leads to lethal chondrodysplasia, in contrast to the mild viable phenotype in mice lacking TGF?RII, demonstrating that ALK5 acts independently of TGF?RII in cartilage. Furthermore, we found that Smad2/3 mutants are viable, demonstrating that ALK5 does not act primarily as a transducer of canonical TGF? signaling in cartilage. Unexpectedly, loss of ALK5 led to a significant increase in BMP output. Biochemical studies pointed to ALK1 as the source of elevated BMP signaling. This was confirmed by our finding that loss of Alk1 rescues the severe chondrodysplasia in Alk5 mutants. Our studies therefore uncover a new mechanism of action for ALK5 as a regulator of BMP output and a previously unknown pathological role for ALK1 in cartilage. We will investigate ALK1 and ALK5 action in growth plate cartilage in Aim 1. We also ablated Alk5 in adult articular cartilage (AC). Mutants develop early onset osteoarthritis (OA)-like pathologies. We will address whether ALK5 acts as a transducer of canonical TGF? pathways and/or as an inhibitor of BMP signaling through ALK1 in AC in Aim 2. Finally, we performed crosslinking, reporter, proximity ligation, and other assays to investigate the basis for the genetic interaction between ALK1 and ALK5. These studies indicate that (a) loss of ALK5 triggers the de novo formation of ALK1/ActRIIB complexes, and (b) provide evidence for ligand- independent BMP signaling through ALK1 in cartilage. Prior in vitro studies have shown that ALK1 is unique among BMP receptors in its ability to activate ligand-independent signaling, but the underlying mechanisms and physiological relevance have never been investigated. We address these unknowns in Aim 3. In summary, these studies demonstrate that the primary role of ALK5 is not to transduce TGF? signals, but rather is to prevent ligand-independent BMP signaling through ALK1 in cartilage. Completion of these studies could lead to a significant revision of our understanding of the role of ALK5 and its critical importance in regulating TGF?/BMP crosstalk in cartilage.